The present invention relates to improved methods and reagents for the production of 1,3-propanediol. In particular, the present invention provides novel thermophilic organisms and thermostable enzymes capable of catalyzing the fermentation of glycerol to 1,3-propanediol. The present invention also relates to methods of isolating such thermophilic organisms, methods of cloning polynucleotides that encode such enzymes, polynucleotides encoding such enzymes, and methods of using such enzymes and organisms for the production of 1,3-propanediol.
1,3-Propanediol is a monomer used in the production of polyester fibers and the manufacture of polyurethanes and cyclic compounds.
A variety of synthetic routes to 1,3-propanediol are known. For example, 1,3-propanediol can be synthesized: (1) by the conversion of ethylene oxide over a catalyst in the presence of phosphine, water, carbon monoxide, hydrogen and an acid; (2) by the catalytic solution phase hydration of acrolein, followed by reduction; or (3) by reacting a hydrocarbon (e.g., glycerol) in the presence of carbon monoxide and hydrogen over catalysts having atoms from group VIII of the periodic table. However, traditional chemical synthesis methods are expensive and generate waste streams containing environmental pollutants, and are thus far from ideal. It would be desirable to develop alternate methods and reagents for the production of 1,3-propanediol that are less expensive and more environmentally friendly.
An alternate approach is to use enzymes, either in vivo (i.e., in a microorganism) or in vitro, to catalyze the fermentation of glycerol to 1,3-propanediol. See, e.g., WO 98/21339, WO 98/21341, and U.S. Pat. Nos. 5,821,092, 5,254,467, 5,633,362 and 5,686,276. Bacterial strains able to produce 1,3-propanediol from glycerol have been found, for example, in the groups Citrobacter, Clostridium, Enterobacter, Ilyobacter, Klebsiella, Lactobacillus, and Pelobacter. These bacteria convert glycerol to 1,3-propanediol by means of a two step, enzyme catalyzed reaction. In the first step, a dehydratase catalyzes the conversion of glycerol to 3-hydroxypropionaldehyde (3-HP) and water (Equation 1). In the second step, 3-HP is reduced to 1,3-propanediol by a NAD+-linked oxidoreductase (Equation 2).
Glycerolxe2x86x923-HP+H2Oxe2x80x83xe2x80x83(Equation 1)
3-HP+NADH+H+xe2x86x921,3-Propanediol+NAD+xe2x80x83xe2x80x83(Equation 2)
The 1,3-propanediol is not metabolized further and, as a result, can accumulate in the media to a high concentration. The overall reaction results in the oxidation of reduced b-nicotinamide adenine dinucleotide (NADH) to nicotinamide adenine dinucleotide (NAD+).
The bioconversion of glycerol to 1,3-propanediol is generally performed under anaerobic conditions using glycerol as the sole carbon source and in the absence of other exogenous reducing equivalent acceptors. In some bacterial strains, e.g., certain strains of Citrobacter, Clostridium, and Klebsiella, a parallel pathway for glycerol metabolism operates which first involves oxidation of glycerol to dihydroxyacetone (DHA) by a NAD+- (or NADP+-) linked glycerol dehydrogenase (Equation 3). The DHA, following phosphorylation to dihydroxyacetone phosphate (DHAP) by a DHA kinase (Equation 4), becomes available for biosynthesis and for supporting ATP generation via, for example, glycolysis.
Glycerol+NAD+xe2x86x92DHA+NADH+H+xe2x80x83xe2x80x83(Equation 3)
DHA+ATPxe2x86x92DHAP+ADPxe2x80x83xe2x80x83(Equation 4)
In contrast to the 1,3-propanediol pathway, this pathway may provide carbon and energy to the cell and produces rather than consumes NADH.
In Klebsiella pneumoniae and Citrobacter freundii, the genes encoding the functionally linked activities of glycerol dehydratase (dhaBCE), 1,3-propanediol oxidoreductase (dhaT), glycerol dehydrogenase (dhaD), and dihydroxyacetone kinase (dhaK) are found in the dha regulon. The dha regulons from Citrobacter and Klebsiella have been expressed in Escherichia coli and have been shown to convert glycerol to 1,3-propanediol (Tong et al., Appl. Environ. Microbiol. 57:3541-46 (1991); Seyfried et al., J. of Bact. 178:5793-96 (1996); Tobimatsu et al., J. Biol. Chem. 271:22352-22357 (1996)).
In view of the potential advantages inherent in the use of biological methods and reagents to produce 1,3-propanediol, their exists a need for the development and identification of novel microorganisms and enzymes capable of converting glycerol and other carbon substrates to 1,3-propanediol having superior characteristics. The present invention satisfies that need by providing superior microorganisms and enzymes, along with methods of identifying other superior microorganisms and enzymes.
The present invention relates to improved methods and reagents for the production of 1,3-propanediol. In particular, the present invention provides novel thermophilic organisms and thermostable enzymes capable of catalyzing the fermentation of glycerol to 1,3-propanediol. The present invention also relates to methods of isolating such thermophilic organisms, methods of cloning polynucleotides that encode such enzymes, polynucleotides encoding such enzymes, and methods of using such enzymes and organisms for the production of 1,3-propanediol.
In one aspect, the invention provides a method of converting glycerol to 1,3-propanediol in a thermophilic organism, the method comprising: providing a thermophilic organism that ferments glycerol to 1,3-propanediol; and culturing the thermophilic organism under conditions such that 1,3-propanediol is produced. In a preferred embodiment, the method further comprises the step of collecting 1,3-propanediol produced by the thermophilic organism. In another preferred embodiment, the thermophilic organism is Caloramator viterbiensis, wherein a thermophilic organism derived from the organism deposited as ATCC designation PTA-584 is particularly preferred.
The invention further provides a method of producing 1,3-propanediol from glycerol, the method comprising: incubating glycerol with a thermostable dehydratase enzyme, thereby converting the glycerol to 3-hydroxypropionaldehyde; and reducing the 3-hydroxypropionaldehyde to 1,3-propanediol. In a preferred embodiment, the reduction of the 3-hydroxypropionaldehyde to 1,3-propanediol is catalyzed by a thermostable 1,3-propanediol oxidoreductase. In another preferred embodiment, the method further comprises the step of collecting 1,3-propanediol. In yet another preferred embodiment, thermostable dehydratase enzyme is derived from a thermophilic organism such as Caloramator viterbiensis, wherein a thermophilic organism derived from the organism deposited as ATCC designation PTA-584 is particularly preferred.
Still another aspect of the invention provides an isolated thermostable glycerol fermentation enzyme that is derived from C. viterbiensis, wherein a thermostable glycerol fermentation enzyme derived from the organism deposited as ATCC designation PTA-584 is particularly preferred. In particular preferred embodiments, the thermostable glycerol fermentation enzyme is a dehydratase, such as glycerol dehydratase, or a NAD+-linked oxidoreductase, such as 1,3-propanediol oxidoreductase. The invention also provides an isolated thermostable glycerol fermentation enzyme that is homologous to a thermostable glycerol fermentation enzyme derived from C. viterbiensis. 
Also provided by the invention is an isolated culture or cell of C. viterbiensis. In a non-limiting embodiment, the genome of the culture or cell is at least 85% identical to the genome of the organisms deposited as ATCC designation PTA-584, preferably 90% identical to the genome of the organisms deposited as ATCC designation PTA-584, more preferably 95% identical to the genome of the organisms deposited as ATCC designation PTA-584, and most preferably at least 99% identical to the genome of the organisms deposited as ATCC designation PTA-584. In another non-limiting embodiment, the 16S rDNA sequence of the culture or cell is at least 95% identical to the 16S rDNA sequence of the organisms deposited as ATCC designation PTA-584, and preferably at least 98% identical to the 16S rDNA sequence of the organisms deposited as ATCC designation PTA584.
In another aspect, the present invention provides a method of cloning a polynucleotide sequence that encodes a thermostable glycerol fermentation enzyme, the method comprising: hybridizing polynucleotide probes homologous to a portion of a known glycerol fermentation enzyme gene to a polynucleotide molecule from an environmental sample suspected of containing a thermophilic organism; and isolating a polynucleotide sequence that binds to at least one polynucleotide probe. In a non-limiting embodiment, the method uses a polymerase chain reaction to amplify the polynucleotide sequence that binds to the polynucleotide probes. In a preferred embodiment, the thermostable glycerol fermentation enzyme is derived from a thermophilic organism identified as fermenting glycerol to 1,3-propanediol, wherein C. viterbiensis is particularly preferred. In another preferred embodiment, the polynucleotide probes are homologous to a portion of a known dhaB gene, wherein probes homologous to the dhaB gene from Klebsiella are particularly preferred.
The invention further provides a method of cloning a polynucleotide sequence that encodes a thermostable glycerol fermentation enzyme, the method comprising: transforming a target organism that cannot grow anaerobically on glycerol with DNA from a thermophilic organism; and identifying those transformed target organisms that contain the polynucleotide sequence that encodes an enzyme that ferments glycerol to 1,3-propanediol by their anaerobic growth on glycerol. In a non-limiting embodiment, the thermostable glycerol fermentation enzyme is derived from a thermophilic organism identified as fermenting glycerol to 1,3-propanediol, such as C. viterbiensis, wherein a thermophilic organism derived from the organism deposited as ATCC designation PTA-584 is particularly preferred.
Still another aspect of the invention provides a method of isolating a thermophilic organism that catalyzes the fermentation of glycerol to 1,3-propanediol, the method comprising: incubating a sample containing thermophilic organisms in media containing glycerol as the primary carbon source; and isolating at least one thermophilic organism that ferments glycerol into 1,3-propanediol. In non-limiting embodiments, the sample is incubated at a temperature in the range of about 40xc2x0 C. to about 100xc2x0 C. and/or under anaerobic conditions. In another non-limiting embodiment, the sample is obtained from a natural source having a temperature of between about ambient to about 100xc2x0 C., and more preferably from about 50xc2x0 to about 100xc2x0 C. In a preferred embodiment, the method further comprises the step of detecting production of 1,3-propanediol and/or acetate by the thermophilic organism.